Crustacea, Decapoda, Brachyura

Journal of the Marine A new species of pea crab from south-western Biological Association of the United Kingdom Europe (Crustacea, Decapoda, Brachyura): species description, geographic distribution cambridge.org/mbi and population structure with an identification key to European Pinnotheridae

Original Article Jose A. Cuesta1, J. E. García Raso2, Pere Abelló3, Elena Marco-Herrero3, Cite this article: Cuesta JA, García Raso JE, Luis Silva4 and Pilar Drake1 Abelló P, Marco-Herrero E, Silva L, Drake P (2019). A new species of pea crab from south- 1 western Europe (Crustacea, Decapoda, Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), Avda. República Saharaui, 2, 11519 Puerto Real, Cádiz, 2 Brachyura): species description, geographic Spain; Departamento de Biología Animal, Universidad de Málaga, Campus de Teatinos s/n, 29006 Málaga, Spain; distribution and population structure with an 3Institut de Ciències del Mar (ICM-CSIC), Passeig Marítim de la Barceloneta 37- 49, 08003 Barcelona, Spain and identification key to European Pinnotheridae. 4Instituto Español de Oceanografía (IEO), Centro Oceanográfico de Cádiz, Puerto Pesquero, Muelle de Levante, Journal of the Marine Biological Association of s/n, PO Box 2609, E-11006 Cádiz, Spain the United Kingdom 99, 1141–1152. https:// doi.org/10.1017/S0025315419000018 Abstract Received: 24 May 2018 After the recent detection, by both morphology and DNA barcodes, of the larval stages of an Revised: 29 November 2018 Accepted: 3 January 2019 unknown species of pea crab (Pinnotheres sp.) in European waters, adults of this crab are herein First published online: 18 February 2019 reported and described as a new species. The current known geographic distribution of the species comprises the Gulf of Cádiz in the eastern Atlantic and the adjacent Mediterranean waters of Key words: the south of the Iberian Peninsula (Alboran Sea), where this crab is well-established inside the Anomia ephippium; Brachyura; distribution; DNA barcoding; European waters; new species; anomiid bivalve Anomia ephippium. In the Gulf of Cádiz, the species displayed a relatively Pinnotheres bicristatus high prevalence: on average, 55.6–77.7%, in A. ephippium samples. The dominant demographic categories of the new species were soft females (61.8–77.0%) with fewer males (17.7–21.10%). Author for correspondence: Most of the host bivalves carried only one crab; in bivalves harbouring two crabs, heterosexual Jose A. Cuesta, E-mail: jose.cuesta@icman. pairs were collected more frequently than expected by chance, which suggests that they could be csic.es mated pairs. A strong correlation between host size and soft female size was found (r = 0.73, P < 0.01) indicating that space availability within hosts seems to be relevant in determining the size of the sedentary phase of the new crab species.

Introduction The Pinnotheridae de Haan, 1833 is a family of brachyuran decapods comprising some 297– 312 species (Ng et al., 2008; Ahyong et al., 2011; Palacios Theil et al., 2016), with several genera and subfamilies that need to be revised (Palacios Theil et al., 2016). Until very recently, four species of the family Pinnotheridae were known to occur in European waters (Becker & Türkay, 2010; Subida et al., 2011), namely Afropinnotheres monodi Manning, 1993, a crab originally from Africa, and the native European Nepinnotheres pinnotheres (Linnaeus, 1758), Pinnotheres pectunculi Hesse, 1872 and P. pisum (Linnaeus, 1767). For these four species there are data available on their distribution, larval stages and DNA markers (Pérez-Miguel et al., 2019). This information allowed the detection by Marco-Herrero et al.(2018) of some pinnotherid larvae in plankton samples (Project ECOBOGUE) from the coast of Doñana National Park (Gulf of Cádiz, SW Spain) that did not belong, either morphologically or according to DNA analyses, to any of the previously known European pinnotherid species. This was considered as an early detection of a new marine invader in European waters. Since DNA analysis of the larvae indicated that the new species was closer to species of the genus Pinnotheres than to any other Pinnotheridae genus, it was provisionally named Pinnotheres sp. (see Marco-Herrero et al., 2018; Pérez-Miguel et al., 2019). Since then, the search was begun for their adult stages, in the context of the AFROBIV project: this is a project to study the pinnotherid species present in the Iberian Peninsula, paying special attention to the effect of A. monodi on their bivalve hosts. Two approaches were © Marine Biological Association of the United Kingdom 2019 adopted in this search: (1) the collection of a large number of individuals of different possible hosts, in the geographic area where larvae appeared; and (2) searching in existing crustacean collections for samples of pinnotherids collected in Iberian waters, for close morphological examination. During a recent and exhaustive sampling of intertidal bivalves all along the Iberian Peninsula coasts, we did not collect any pinnotherid adults of any unidentifiable species (Pérez-Miguel et al., 2019), and we therefore concluded that adult individuals of Pinnotheres sp. would probably be hosted only by subtidal species. A reexamination of the pea crabs (by morphology and DNA barcodes) deposited in the Biological Reference Collections (CBR) at the Institut de Ciències del Mar (ICM-CSIC), resulted in their classification to three out the four known European species, namely N. pinnotheres, P. pectunculi and P. pisum. However, when reviewing specimens from previous studies by Manjón-Cabeza &

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Table 1. Locations, surveys and dates in which the pea crab Pinnotheres bicristatus sp. nov. has been collected

Sampling site Code Coordinates Depth (m) Survey Date

Doñana N.P. coast A1 36°57.5′N 6°32.5′W 11 ECOBOGUE (MT10) 14 November 2013 Doñana N.P. coast A2 36°51.5′N 6°29.0′W 14 ECOBOGUE (PD10) 14 November 2013 Cádiz Bay offshore A3 36°34.8′N 6°27.5′W28–30 ARSA 0318 (L24) 25 February 2018 Cádiz Bay offshore A4 36°34.1′N 6°26.6′W23–27 ARSA 1117 (L102) 30 October 2017 Cádiz Bay offshore A5 36°32.7′N 6°23.7′W20–21 ARSA 1117 (L101) 30 October 2017 Cádiz Bay offshore A6 36°32.7′N 6°23.6′W19–21 ARSA 1117 (L1) 30 October 2017 Barbate offshore A7 36°13.2′N 6°14.0′W 52 ARSA 0318 (L1) 19 February 2018 Barbate offshore A8 36°09.7′N 5°53.6′W15–18 Project PB92–0415 22 July 1994 Barbate offshore A8 36°09.7′N 5°53.6′W15–18 Project PB92–0415 17 March 1995 Estepona offshore M1 36°20.3′N 5°12.9′W 47 MEDITS_ES 2017 (L007) 25 April 2017

Codes of sampling sites as in Figure 1.

García Raso (1998) one ovigerous female captured in Barbate inspected for the presence of pea crabs, which were preserved in (Gulf of Cádiz) was identified as Pinnotheres sp. based on DNA 80% ethanol. The A. ephippium collected during the ARSA barcodes, and it showed a clearly different morphology from 1117 and ARSA 0318 (hosting 98.4% of crabs examined) were fro- that of other European crab species. Also, a soft female living zen in the ship, and later examined in the laboratory, where they inside the common saddle oyster Anomia ephippium Linnaeus, were measured and inspected for the presence of crabs. For A. 1758 (Anomiidae Rafinesque, 1815) had been collected in the ephippium from the ARSA cruises, the maximal shell lengths same area in 1995 (García Raso & Manjón-Cabeza, 2002), and along the anterior-posterior and dorsal-ventral axes of the shell although it was initially identified as P. pisum, it was actually were measured to the nearest 0.1 mm with a dial caliper (Tesa close to the above-mentioned ovigerous female of Pinnotheres Cal IP65). Due to the variability in the shell shape, the shell sp. Given these findings, we then focused on obtaining samples size of A. ephippium was estimated as the mean values of both of Anomia ephippium from the same area to search for more spe- measures. cimens and confirm the habitat and distribution of this species. All crabs found within each individual host were preserved In this study, we analyse by morphology and at the molecular and stored in 80% ethanol for further demographic observations. level the specimens from Barbate as well as the pea crabs hosted The carapace width (CW = maximum cephalothorax width) and by bivalves collected in two oceanographic surveys conducted in carapace length (CL = distance from the rostrum to the posterior 2017 (ARSA 1117 and MEDITS_ES2017) and another survey in margin of the carapace) of each crab was measured to the 2018 (ARSA 0318), paying special attention to A. ephippium. nearest 0.01 mm under a stereomicroscope provided with a cali- All of these specimens clearly belong to an undescribed species brated ocular micrometer. The wet weight (WW) was estimated of the genus Pinnotheres Bosc, 1801. This new species is with a digital balance (precision 0.1 mg). Crabs were sexed described, figured and compared with congeners, and a key for based on the presence (male) or absence (female) of gonopods. European Pinnotheridae is provided. Furthermore, each female crab was classified either as hard or soft by considering its pleonal morphology, cephalothorax consistency and shape, and pleopods morphology: hard females Materials and methods show a flattened and rounded cephalothorax and swimming Material examined legs (similar to those of males), whereas the body of soft females is mostly sub-trapezoidal and lacks swimming legs. Lastly, soft The Pinnotheres bicristatus sp. nov. individuals examined in this females carrying eggs (embryos) were classified as ovigerous study correspond to those hosted by subtidal bivalves collected females. Gonadal development of soft females was considered. by the authors during different fishery research cruises along The drawings were made using a camera lucida and the photo- the Atlantic and Mediterranean coasts of Andalusia (South of graphs using a Nikon digital camera DXM1200F, both mounted Iberian Peninsula). Samples of Anomia ephippium were obtained on stereomicroscopes. Other abbreviations used: MXP3, third from the ARSA 1117, ARSA 0318 and MEDITS_ES 2017 surveys maxilliped; P1–P5, pereiopods 1–5(P1– cheliped, P2–P5 – walk- using bottom trawl gears; an additional pea crab was collected ing legs); IEO-CD, Decapod Crustacean Collection from Cadiz inside A. ephippium, and another was collected free-living in Oceanographic Centre; CBR, Biological Reference Collections. bivalve samples from offshore the Barbate coast (Project PB92-0415) with a dredge (Manjón-Cabeza & García Raso, 1998; García Raso & Manjón Cabeza, 2002). The coordinates of Data treatment of the Gulf of Cádiz population all sampling sites in which Pinnotheres bicristatus sp. nov. has been collected are shown in Table 1, and a map showing the pos- For each of the two ARSA surveys, the host-size related prevalence ition of the sampling stations is displayed in Figure 1. The of Pinnotheres bicristatus sp. nov. in the host Anomia ephippium description of the new species in this paper is only by two of was analysed by estimating the number of crabs collected for each the authors, J.E. García Raso and J.A. Cuesta. 5 mm size group of the host, as well as by representing the cumulative number of A. ephippium in size-increasing order vs the cumulative number of crabs retrieved from them. In addition, Collection and measurement of hosts and crabs for each demographic category of crabs, the relationship between The specimens of Anomia ephippium collected during the host size and crab size was assessed by regression analysis by fit- MEDITS_ES 2017 survey were opened on board the ship and ting data to a linear model. Similarly, the relationship between

edited using the software Chromas version 2.0. The obtained final DNA sequences were compared with those from Pinnother- idae available in GenBank database. An evolutionary distances analysis was carried out in MEGA6 (Tamura et al., 2013). A concatenated alignment of the 16S, Cox1 and H3 sequences was built using the sequences obtained in the present study from specimens of Pinnotheres bicristatus sp. nov., as well as the sequences of A. monodi, N. pinnotheres, P. pectunculi, P. pisum, Pinnotheres sp. (larvae), Orthotheres barbatus (Desbonne, 1867) and Zaops ostreus (Say, 1817) downloaded from GenBank (http://www.ncbi.nlm.nih.gov). The phylogeny reconstruction analyses were inferred from neighbour-joining using the p-distance method. The nodal confidence of the obtained topologies was assessed via 2000 bootstrap replicates.

Results Geographic distribution and host of Pinnotheres bicristatus sp. nov. The known distribution of P. bicristatus sp. nov. is, so far, the Gulf of Cádiz (NE Atlantic) and western Alboran Sea (SW Mediterranean) coasts (Figure 1). In this study, we have examined one ovigerous female collected free-living in bivalve samples in Barbate offshore (Gulf of Cádiz) and 328 individuals hosted by Anomia ephippium: 325 specimens collected along the coast of the Gulf of Cádiz and 3 specimens collected in the Alboran Sea (Table 2).

Fig. 1. Map showing sampling sites where Pinnotheres bicristatus sp. nov. was col- Gulf of Cádiz population lected. ECOBOGUE 2013: A1, A2, Doñana National Park coast; ARSA 2017: A4, A5, A6 offshore Bay of Cadiz; ARSA 2018: A3, offshore Bay of Cadiz, A7, offshore Crab prevalence and size and sex structure Barbate coast; Project PB92–0415: A8, Barbate coast; MEDITS_ES 2017: M1, offshore In the Gulf of Cádiz, the mean prevalence by Pinnotheres bicris- Estepona. tatus sp. nov. in its unique known host, Anomia ephippium,was 55.6% (48.15–66.67%) in October 2017 and 77.7% (67.2–88.2%) in February 2018. crab carapace width and crab wet weight was assessed by regres- Neither megalopae nor indeterminate (small individuals with- sion analysis by fitting data to power regression models. out sexual characteristics) individuals of P. bicristatus sp. nov. Differences between the estimated slopes of regression lines or were found inside the host. The three demographic categories between the exponents of power models were tested by using observed were males, hard females and soft females (Table 2, ’ Student s t-tests. Figure 2). In P. bicristatus sp. nov. population from the Gulf of To test whether crabs tend to live solitarily, in pairs or aggre- Cádiz (98.4% of the crabs examined), the dominant category gated in higher numbers inside the host, the frequency of occur- was soft females (77.0% in October 2017 and 61.8% in February rence of A. ephippium without crabs and with different numbers 2018), followed by males (17.7% in October 2017 and 21.1% in of crabs was compared with the expected frequencies under a ran- February 2018) and hard females (5.3% in October 2017 and dom distribution (binomial distribution) and differences tested by 17.1% in February 2018). The carapace width (and wet weight) χ2 a test. Similarly, the observed proportion of males to females of crabs ranged from 2.0 (4.0 mg) to 10.4 mm (354.9 mg), with χ2 was tested for deviation from a 1:1 sex ratio by using a test. soft females showing the largest average size of 7.2 ± 0.1 mm Besides, the observed sexual composition of crab pairs sharing a (155.4 ± 5.7 mg) in October 2017 and 5.9 ± 0.1 mm (112.0 ± single host individual was compared with the expected frequen- 7.3 mg) in February 2018, followed by males with a mean size cies of heterosexual and homosexual pairs in a random distribu- of 3.9 ± 0.1 mm (55.8 ± 2.4 mg) in October 2017 and 3.5 ± χ2 tion by using test. 0.1 mm (25.3 ± 2.7 mg) in February 2018, and hard females A 5% significance level (P = 0.05) was considered for all statis- with mean size of 3.1 ± 0.1 mm (18.1 ± 1.7 mg) in October 2017 tical tests carried out in this study (Zar, 2010). and 3.1 ± 0.2 mm (16.0 ± 2.4 mg) in February 2018. Most soft females collected in both surveys showed well developed reddish gonads (98.7% in October and 87.2% in February) and the smal- Pinnotheres bicristatus sp. nov. identification by DNA lest female with this characteristic had a carapace width of 3.5 mm barcodes (February 2018). Total genomic DNA was extracted from pereiopod muscle tissue, The sex-ratio (female/male) was: 4.0 in October 2017, which following a modified Chelex 10% protocol by Estoup et al.(1996). differed significantly from a 1:1 sex ratio (χ2 = 103.2, P < 0.01), Partial sequences of the mitochondrial 16S and Cox1, and nuclear and 3.8 in February 2018, which also differed significantly from H3 genes were amplified. Cycling conditions of the polymerase a 1:1 sex ratio (χ2 = 13.9, P < 0.01). Irrespective of the sampling chain reaction (PCR), length of the sequences obtained, and pri- date and demographic category, there was a significant (P < mers used for each gene are the same as in Pérez-Miguel et al. 0.01) power relationship between carapace width and wet weight (2019). PCR products were sent to Stab-Vida laboratories to be (Figure 2). The exponent of fitted models for males (b = 2.979) purified and then bidirectionally sequenced. Sequences were was very close to 3 (t-student = 0.1; P > 0.05), whereas those of

Table 2. Characteristics of pea crabs, Pinnotheres bicristatus sp. nov., examined in this study

Host Site Sexual category No. of crabs Mean CW ± SE CW range Mean WW±SE WW range

A. ephippium A4, A5, A6 Fh 13 3.1 ± 0.1 2.3–3.5 18.1 ± 1.7 1.4–261.0 Fr 191 7.2 ± 0.1 4.3–10.4 155.4 ± 5.7 26.1–354.9 M 44 3.9 ± 0.1 2.3–5.0 55.8 ± 2.4 4.0–67.6 A3, A7 Fh 13 3.1 ± 0.2 2.0–4.1 16.0 ± 2.4 6.9–46.0 Fr 47 5.9 ± 0.1 3.9–7.5 112.0 ± 7.3 15.8–223.6 M 16 3.5 ± 0.1 2.8–4.3 25.3 ± 2.7 14.6–38.6 A8 Fr 1 6.5 – 123.6 – M1 Fr 1 7.1 – 124.8 – Fo 1 7.2 – 195.9 – M 1 3.9 – 23.4 – Not known A8 Fo 1 8.2 226.0 –

CW, crab carapace width (mm); WW, crab wet weight (mg); Fh, hard females; Fr, reproductive females; M, males. Codes and locations of sampling sites (A3 to A8, and M1) as in Figure 1.

soft females (b = 2.597) and hard females (b = 2.519) were lower females (r = 0.73; P < 0.01) and, to lesser extent, males (r = 0.49; than 3 although this difference was statistically significant for P < 0.01). Nevertheless, soft females hosted by A. ephippium of soft females (t =4.3; P < 0.01) but not for hard females (t = 1.0; a specific size were larger than males hosted by A. ephippium of P > 0.05). the same size (Figure 3B).

Interactions between crabs Pinnotheres bicristatus sp. nov. identification by molecular The number of crabs per host varied between 0 and 2, with 96.7% analysis of Anomia ephippium with crabs hosting a single individual in October 2017 and 73.3% in February 2018 (Figure 3A). Thus, All the sequences obtained for the three genes (mithocondrial 16S crab distribution among hosts did not display a random pattern: and Cox1, and nuclear H3) fit 99–100% with those of Pinnotheres there were more bivalves harbouring one crab and fewer bivalves sp. larvae (zoeae and megalopa) deposited in GenBank under the without crabs or harbouring two crabs, than those expected by accession codes MF069149 and MF069150 (16S), MF134396 chance alone. The observed departure from a random pattern (Cox1) and MF138913 (H3) obtained by Marco-Herrero et al. was statistically significant in October 2017 (χ2 = 54.3, P < 0.01) (2018) and Pérez-Miguel et al.(2019). In the final tree, all sequences and February 2018 (χ2 = 10.7, P < 0.01). A total of six A. ephip- of Pinnotheres bicristatus sp. nov. (adults and larvae) clustered pium harbouring heterosexual pairs of crabs were collected in together within a clade, separate from the other European October 2017 (3.3% of infested hosts); whereas 12 A. ephippium Pinnotheres species (P. pisum and P. pectunculi)(Figure 4). with crab pairs (26.7%) were collected in February 2018: 10 heterosexual pairs, one 2-males pair and one 2-females pair. Species description Considering the sex ratio observed in each survey (Table 2), there were more heterosexual pairs than expected by chance Systematic alone both in October 2017 (χ2 = 9.2; P < 0.01) and in February Order DECAPODA Latreille, 1802 2018 (χ2 = 11.3; P < 0.01). One male and one soft female was Infraorder BRACHYURA Latreille, 1802 the combination observed in 55% of heterosexual pairs, whereas Family PINNOTHERIDAE De Haan, 1833 one male and one hard female combination was observed in Genus Pinnotheres Bosc, 1801 45% of them. Pinnotheres bicristatus sp. nov. García Raso & Cuesta (Figures 5–8 & 9G) Host size effects Pinnotheres sp.: Manjón-Cabeza & García Raso (1998), oviger- The collected Anomia ephippium in October 2017 survey had a ous female; Pinnotheres pisum: García Raso & Manjón-Cabeza mean size (±SE) of 40.4 (±0.4) mm and ranged between (2002), soft female; Pinnotheres sp.: Marco-Herrero et al. 16.6 mm and 55.3 mm, whereas those of February 2018 had a (2018), zoea II, zoea III, zoea IV and megalopa. mean size of 35.6 (±0.8) mm ranging from 15.2 to 52.0; similarly, the mean size of specimens hosting pea crabs was 40.5 (±0.5) mm Material examined in October 2017 and of 36.0 (±0.9) mm in February 2018. Holotype: ♂ (4.4 mm CW) (IEO-CD-ICMAN/2419, GenBank A higher number of crabs was collected in the most frequent accession numbers MK415384, MK426944 and MK468907) in size classes of A. ephippium in both surveys. This homogeneous Anomia ephippium Linnaeus, 1758 (Bivalvia, Anomiidae), prevalence of crabs throughout the host size range was evidenced 36°34.1′N 6°26.6′W, Gulf of Cádiz, 23–27 m depth, detritic bot- by an increase of the cumulative number of crabs collected pro- tom, coll. Luis Silva (ARSA cruise 1117, 30 October 2017). portional to that of the cumulative number of hosts when the lat- Paratypes: in Anomia ephippium Linnaeus, 1758 (Bivalvia, ter was estimated by ordering hosts by increasing size (Figure 3A). Anomiidae), 36°34.1′N 6°26.6′W, Gulf of Cádiz, 23–27 m depth, det- For crabs collected in the 2017 and 2018 surveys, there was no ritic bottom, coll. Luis Silva (ARSA cruise 1117, 30 October 2017): significant correlation between hard female sizes and those of 1 ♀ (hard) (3.27 mm CW) (IEO-CD-ICMAN/2420), 1♂ and 1♀ their hosts (r = 0.07; P > 0.05), whereas a significant positive cor- (soft) (3.8 and 7.5 mm CW) (IEO-CD-ICMAN/2421–2422), 1 ♀ relation between host and crab sizes was observed for both soft (soft) (7.35 mm CW) (IEO-CD-ICMAN/2423); 1 ♀ (soft)

Fig. 2. Relationship between Pinnotheres bicristatus sp. nov. carapace width and wet weight for each demographic category, and percentage represented by each category (pie chart) in October 2017 and February 2018 surveys in Gulf of Cadiz.

(7.8 mm CW) (IEO-CD-ICMAN/2424), 1 ♀ (soft) (7.50 mm CW) reaching end of propodus. First thoracic sternite with anterior (IEO-CD-ICMAN/2425), 1 ♂ and 1 ♀ (soft) (3.8 and 5.08 mm CW) border concave. (IEO-CD-ICMAN/2426–2427), 1 ♀ (7.88 mm CW) (IEO-CD- Chelipeds (Figures 5C & 6A) similar. Palm (pal) slightly − ICMAN/2428), 1 ♀ (soft) (8.03 mm CW) (IEO-CD-ICMAN/2429), longer than dactylus (d) (pal d 1 = 1.05), relationship length/ 1 ♀ (soft) (6.89 mm CW) (IEO-CD-ICMAN/2430), 1 ♀(soft) width of palm = 1.1. Dactylus with well-developed tooth basally, (8.26 mm CW) (IEO-CD-ICMAN/2431), 1 ♀ (soft) (5.68 mm smaller tooth present anteriorly; cutting edge of fixed finger of CW) (IEO-CD-ICMAN/2432), 1 ♀ (soft) (7.42 mm CW) (IEO- propodus with large tooth (in anterior position to that of mobile CD-ICMAN/2433), 1 ♂ (3.6 mm CW) (IEO-CD-ICMAN/2434), finger), with about 15 short thick setae between apex of tooth. 1 ♀ (soft) (5.76 mm CW) (IEO-CD-ICMAN/2435), 1 ♂ (3.8 mm Tips of fingers curved. CW) (IEO-CD-ICMAN/2436). In Anomia ephippium, 36°34.8′N Walking legs (Figure 5F–I, pereiopods 2–5) slender. P3, P4 6°27.5′W, Gulf of Cádiz, Spain, 28–30 m depth, detritic bottom, longer than P2, P5. P3 slightly longer than P4. Relative length coll. Luis Silva (ARSA expedition 0318, 25 February 2018): 1♀ P3 > P4 > P2 > P5. Merus of P2–P5 with short setae on dorsal (hard) (2.59 mm CW) (IEO-CD-ICMAN/2437), 1 ♂ and 1 ♀ and ventral sides. P3 and P4 with long swimming setae, in carpus (hard) (3.6 and 2.9 mm CW) (IEO-CD-ICMAN/2438–2439). In (on lateral outer side), propodus and dactylus (on dorsolateral Barbate offshore, 36°09.7′N 5°53.6′W, free in bivalve samples of det- outer, and ventral sides), and some on merus of P5 (dorsodistal ritic bottom, 15–18 m depth, 22 July 1994, coll. Manjón-Cabeza and and ventral parts), carpus and propodus (dorsal and ventral García Raso (1988, as Pinnotheres sp.): 1 ovigerous ♀ (8.2 mm CW) parts). Without long swimming setae on P2 (Figure 5F). (IEO-CD-ICMAN/2440, GenBank accession numbers MK426940 Relative lengths of merus are: P3 > P4 > P2 > P5; relationships and MK468903). In Anomia ephippium, Barbate offshore, 36° length/width of merus leg P2 to P5 are 3.1, 3.1, 3.6 and 2.6 09.7′N 5°53.6′W, detritic bottom, 15–18 m depth, 17 March 1995, respectively. Proportion of length of merus/length of dactylus of coll. García Raso and Manjón Cabeza (2002, as Pinnotheres pisum): P2 to P5 are 2.7, 2.4, 2.4 and 2.0 respectively. Propodus relative 1 ♀ (soft) (6.5 mm CW) (IEO-CD-ICMAN/2441). In Anomia lengths are: P3 > P4 > P2 > P5. Dactylus of P2–P5 almost straight, ephippium, Estepona offshore, 36°20.3′N 5°12.9′W, 47 m with long curved distal part (claw), with small spiniform setae on depth, coll. Pere Abelló (MEDITS_ES 2017, 25 April 2017): 1 ♂ ventral concave faces, longer in P5. Dactylus of P3 and P4 longer (3.9 mm CW) (ICMD002583, GenBank accession numbers than others, subequal in length, P3 ≥ P4 > P2 > P5 (Figure 5F–I). MK415382, MK426942 and MK468905), 1 ♀ (soft) (7.1 mm Male pleopod curving regularly, with apex narrowing progres- CW) (ICMD002584, GenBank accession numbers MK415381, sively from base to tip (Figure 5D), with abundant setae from base MK426941 and MK468904), 1 ovigerous ♀ (7.2 mm CW) to apex, especially on concave face. (ICMD002585, GenBank accession numbers MK415383, MK426943 Pleon (Figure 5E) triangular, gradually narrowing, with 6 and MK468906). somites and telson all free with rectilinear distal margin.

Description of male. Carapace (Figures 5A & 6A–C) circular (CL/ Description of hard female. These small females have long swim- CW = 0.99). Minimum and maximum width: 3.64 and 4.4 mm, ming setae on their pereiopods, similar to those of males. The gen- respectively. Frontal margin projected anteriorly with dense eral morphology is also very similar to that of males; the carapace brush of short setae medially. Dorsoanterolaterally, on both is circular, with the same characteristic dorsoanterolateral tufts of sides of carapace, characteristic dense group of short and thick short setae (Figures 7A & 8A). Also, the morphology of chelipeds setae (more than 30) present, superficially resembling tubercles is similar to that of males. Relative length of walking legs is: P3 > (Figure 6B). Eyes clearly visible in dorsal view. Antenna short, P4 > P2 > P5. The dactyli of P3 and P4 are the longest and both are with large basal segment followed by 4 segments, and with 2 or of similar lengths. The length/width relationships of the propodus – 3 terminal setae on distal segment. (P2 P5) are: 2.18, 2.42, 2.33 and 1.73. Propodus/dactylus length – Third maxilliped (Figure 5B) with ischium and merus indistin- relationships (P2 P5) are: 1.60, 1.45, 1.56 and 1.33. Pleon only guishably fused, inner, outer margins with distal obtuse angle; slightly wider than that of adult males (Figure 8B). palp 3-segmented with tuft of long simple setae from tip; propodus longer than carpus, dactylus inserted underneath propodus, Description of soft females. Carapace (Figures 7B & 8C, E) subtra- on proximal half of ventral margin of propodus, apex not pezoidal, wider than long (CL/CW = 0.76 to 0.81). Minimum and

Fig. 4. Topology of neighbour-joining tree based on concatenated 16S and Cox1 mtDNA and H3 nuclear gene sequences, showing inferred phylogenetic relationships within European Pinnotheridae, with American Zaops ostreus and Orthotheres barbatus as outgroups. Numbers close to nodes indicate bootstrap support (only values above 80% shown).

relative lengths are: P3 > P4 > P2 > P5. Dactylus of P2–P5 almost straight with curved distal part, with continuous row of tiny spines on ventral face of P2 and P4 (not in P3 and P5). Dactylus of P3 longer than rest (P3 > P4 > P2 > P5) (Figures 7B, G & 8C–E); more clearly in large specimens (Figures 7B, G & 8C, D). Dactylus of P5 narrower than on others. Relationships between merus (m), propodus (p) and dactylus (d) lengths − of each P2 to P5 are as follow: m d 1 = 2.5, 2.0, 2.3 and − 1.6; p d 1 = 1.5, 1.4, 1.7 and 1.5. Pleon, with 6 somites, very broad (Figures 7C & 8D); telson well separated with rectilinear distal margin, covering almost completely ventral side, even covering almost completely the third maxillipeds (in large females), with setose margin and Fig. 3. Relationship between host shell size and Pinnotheres bicristatus sp. nov. popu- smooth outer surface. lation from Gulf of Cadiz. (A) Cumulative number of Anomia ephippium in increasing Etymology: The specific name bicristatus is an adjective (gen- size order vs cumulative number of pea crabs retrieved from them (plot on left top der masculine as Pinnotheres) referring to the two ‘crests’ or corner) in October 2017 (solid line) and February 2018 (dotted line) surveys; number of pea crabs by host (bar plot on right top corner) and pea crab prevalence by host dense tufts of setae located dorsoanterolaterally on the carapace size-groups (main bar plot) in October 2017(each left bar) and February 2018 (each of males and hard females. right bar) surveys. (B) Host shell size vs carapace width of pea crab for each crab Habitat, host: All specimens collected were found inside demographic category. ** and *, significance level of correlation coefficient of P < Anomia ephippium collected between 11 and 47 m depth, on det- 0.01 and P < 0.05, respectively. ritic bottoms. Colouration in life: Males and hard females carapace with maximum width of carapace: 5.08 and 8.2 mm, respectively. yellow-reddish colour with dark spots and with a characteristic Dorsal surface smooth, without dorsoanterolateral tufts of short pattern; it resembles a palm tree (Figures 6A & 8A), with a well- ‘ ’ setae. Front almost rectilinear, not projected, without setae defined central trunk and two pairs of lateral projections as ‘ ’ ‘ ’ (slightly convex in smallest soft females, AR-156: 5.08 mm leaves , the posterior ones separated from this trunk . Behind, CW). Small eyes, hardly visible in dorsal view. Antenna short, there are two well-defined rounded areas. The whole outline of with large basal segment followed by 4 segments, and with 2 or this figure is reddish. Soft females with a pale-yellow colour: 3 terminal setae on distal segment. when females present developed gonads, they are reddish. Third maxilliped (Figure 7D) obliquely positioned on buccal cavity, not different from P. pisum (see Figure 5C in Becker & Discussion Türkay, 2010), ischium and merus indistinguishably fused, inner, outer margins with distal obtuse angle; palp 3-segmented Morphology with tuft of long simple setae at tip; propodus longer than carpus; Taxonomically, within the genus Pinnotheres the species richness dactylus inserted underneath propodus, almost subterminal, at or is high, with a total of 52 species (WoRMS 2018) or 61 species about half length of ventral margin of propodus, apex reaches to (Palacios Theil et al., 2016), but only two species are known in or does not reach end of propodus. the area studied: P. pisum and P. pectunculi. In the past this Chelipeds (Figure 7E) similar. Palm longer than dactylus (pal genus was the most specious of the Pinnotheridae, but after sev- − d 1 = 1.2–1.3), relationship length/width of palm = 1.2–1.3 (nar- eral studies, especially those of Manning (1993a, 1993b) and rower than that of males and hard females). Dactylus with well- Campos (1989, 1993, 2002), new genera have been recognised developed tooth basally, another on cutting edge of fixed finger for many former Pinnotheres species. In contrast, no new species of propodus. of Pinnotheres s. str. have been described since Pinnotheres tai- Walking legs (Figures 7F–I) slender. P3 longer than rest. Total chungae Sakai, 2000. relative length is: P3 > P4 > P2 > P5. Merus relative length: P3 > P4 ≈ P2 > P5. Length/width relationships of merus (P2–P5) are Morphological differences with other European pinnotherids. In 4.8, 5.1, 4.1 and 3.3 (P3 > P2 > P4 > P5), respectively. Propodus European waters pinnotherid species have been known since

Fig. 6. Pinnotheres bicristatus sp. nov. Holotype male IEO-CD-ICMAN/2419 (A, C) dorsal view, showing colour pattern; (B) detail of dorsoanterolateral carapace region with tufts of short curved setae. Fig. 5. Pinnotheres bicristatus sp. nov. Holotype male IEO-CD-ICMAN/2419 (4.4 mm carapace width). (A) Carapace, dorsal view and figure pattern; (B) third maxilliped, outer and inner views; (C) right cheliped, outer view; (D) male first gonopod; (E) pleon, outer view; (F–I) outer views of the second to fifth right pereiopods. Scale junior synonyms of N. pinnotheres, based on the original descrip- – bars: (A, C), (E I), 1.0 mm; (B, D), 0.5 mm. tions and figures (because there is no type material extant) and together with the fact that the hosts of both species are ascidians. Based on the same evidence and the previous paper by Gourret Greek and Roman times (see Becker, 2010), although no descrip- (1887), we have also concluded that they are junior synonyms tions of species were made until the 1700s. According to Ng et al. of N. pinnotheres, and we are confident neither can be P. bicrista- (2008) there are several old studies that were overlooked in the tus sp. nov. first description of Pinnotheres pisum (as Cancer pisum)by When comparing P. bicristatus sp. nov. with the recognized Linnaeus (1767) and subsequent studies. Scopoli (1763) described species of European pinnotherids, males and hard females from Cancer nutrix as a crab living inside Ostrea edulis from the all those species can be easily differentiated by two peculiar char- Adriatic Sea. As Ng et al.(2008) pointed out, de Wulfen (1791) acteristics on the cephalothorax: (a) a pair of dorsoanterolateral also described, from the Adriatic coasts, Cancer minutus, a species tufts of curved setae (short and very close to each other) that very similar to Cancer nutrix and living inside O. edulis, but also resemble two tubercles (Figures 5A, 6B & 7A), not described in in other bivalves. According to the very brief descriptions given the previous known European species or in other species for by these authors and considering that there are no type specimens which we have evidence; and (b) the orange-reddish ‘palm tree’ extant available for checking, we believe that both taxa belong to marking covering the dorsal surface (Figures 5A, 6A, C, 7A & P. pisum, mainly because this species is present in that part of the 8A). Concerning this latter feature, males of P. pisum show a Mediterranean Sea and is known to inhabit several different spe- somewhat similar, although different, marking in which the cies of hosts, including O. edulis. At present, the complete distri- ‘trunk of the palm tree’ is missing and the colour is yellow- bution of Pinnotheres bicristatus sp. nov. is unknown and it orangish (see pictures in the Crustikon – Crustacean photo- cannot be excluded that this new species could be one of those graphic website – Tromsø Museum, University of Tromsø, described by Scopoli (1763) and de Wulfen (1791). We have, http://archive.li/rHrj6, by Cédric d’Udekem d’Acoz; and however, found only P. bicristatus sp. nov. inside Anomia ephip- Figure 1B in Becker & Türkay, 2010). pium, and not in any other bivalve species. Pinnotheres bicristatus sp. nov. can be differentiated from the Assuming that C. nutrix can be assigned to P. pisum, and that African species Afropinnotheres monodi by the morphology of Scopoli’s description in 1763 has priority with respect to that of the third maxilliped: in A. monodi the propodus is shorter than Linnaeus (1767), the correct name for P. pisum would be the carpus (this is not the case in the genus Pinnotheres and Pinnotheres nutrix (Scopoli, 1763). Prior to being more conclu- Nepinnotheres), and the dactylus extends well beyond the end of sive, as suggested by Ng et al.(2008), it could be necessary to the propodus (see A. monodi Figure 1A, in Manning, 1993a), assign neotypes to these species by collecting fresh specimens while these features are not present in the rest of European species hosted by O. edulis from the Adriatic Sea and also take into (at most, the dactylus extends only very slightly in N. pinnotheres; account that Ŝtevčić (1990) also mentioned N. pinnotheres inside see Figure 3C in Becker & Türkay, 2010). Also, in A. monodi, the Ostrea edulis in this region. This is, however, beyond the scope of dorsal surface of the carapace in males and hard females is finely the present study. pubescent. Two other species have been described from European waters, Soft females of the new species and those of Nepinnotheres Pinnotheres ascidicola Hesse, 1872 and P. marioni Gourret, 1888. pinnotheres can be differentiated easily by the apex of the dactylus Recently, Becker & Türkay (2010) made a detailed study of of the third maxilliped, which reaches or exceeds the distal part of European pinnotherids and concluded that both species are the propodus in N. pinnotheres (see Figure 24B in Manning,

Fig. 8. Pinnotheres bicristatus sp. nov. (A, B) Paratype hard female IEO-CD-ICMAN/ 2420, habitus: (A) dorsal view; (B) ventral view. (C, D) Paratype ovigerous female IEO-CD-ICMAN/2440, habitus, dorsal and ventral views (left and right respectively); (E) Paratype soft female (7.88 mm carapace width), IEO-CD-ICMAN/2428, from Gulf Fig. 7. Pinnotheres bicristatus sp. nov. (A) Paratype hard female (3.27 mm carapace of Cadiz, in dorsal view (right) and detail of walking left legs in ventral view (left). width) IEO-CD-ICMAN/2420, dorsal view; (B–I) Paratype ovigerous female (8.2 mm carapace width), IEO-CD-ICMAN/2440, from Barbate (setae not represented): (B) carapace in dorsal view; (C) ventral view; (D) third right maxilliped (outer view); (E) chela of first left pereiopod (outer view); (F–I) outer view of second to fifth left pereiopod. Scale bars: (A–C), (E–I), 1.0 mm; (D), 0.5 mm. Becker & Türkay, 2010), not subtrapezoidal as in the new species (Figures 7B & 8C, E).

1993a; Figure 3C in Becker & Türkay, 2010), but not in P. bicris- Identification key for European Pinnotheridae. tatus sp. nov. (Figure 7D). The carapace morphology is subglobular, with width greater than length, in N. pinnotheres, whereas it 1. Mxp3 with propodus conical, tapering distally, shorter is subtrapezoidal in P. bicristatus. However, both species possess a than carpus. Dactylus inserted at base of ventral margin of broad triangular tooth at about mid-length of the fixed finger of propodus, overreaching end of propodus ...... the chelipeds (which does not exist, or else is very small, in P...... Afropinnotheres monodi pisum), and the dactylus lengths of P5 are more than half the length of their propodus. However, in the new species, the dacty- Mxp3 with propodus oval or spatulate, longer than carpus. lus of P3 is clearly longer than those of the rest of its walking legs, Dactylus inserted at mid-length or at base of ventral margin of a difference especially pronounced in large females (Figures 7B, G propodus but apex not extending to end of propodus ...... 2 & 8C, D). Soft females of Pinnotheres pectunculi have a subtrapezoidal 2. Carapace of hard specimens (males and females) with dor- carapace with an approximately rectilinear front that is not pro- soanterolateral tuft of setae. Soft females with subtrapezoidal jected (see Figure 25 in d’Udekem d’Acoz, 1989 and Figure 9A, carapace, dactylus of P3 longer than others, dactylus of P5 nar- B in the present work) as in P. bicristatus sp. nov. (Figures 7B rower, with a relation propodus/dactylus length of & 8C, E) (in N. pinnotheres the frontal area is a little projected, 1.5 ...... Pinnotheres bicristatus sp. nov. bilobed by a median incision, whereas in P. pisum it is slightly rounded). They can also be easily distinguished by the dactyli Carapace of hard specimens (males and females) without dor- of the walking legs, which are very different. Thus, in P. pectunculi soanterolateral tuft of setae. Soft females with subtrapezoidal or they are very short (d’Udekem d’Acoz, 1989)(Figure 9A, B) (even subglobular-rounded carapace, dactylus of P2-P5 more or less shorter than those of P. pisum), while they are long in the new similar in length ...... 3 species, especially in large soft females, whose legs are longer, and thinner (stylized) than those of hard individuals. 3. Carapace and pereiopods covering with tomentum. P2–P5 With regards to morphology, Pinnotheres pisum is the species with long dactylus, that of P5 slightly longer than others, closest to P. bicristatus sp. nov. They can be separated by the rela- approximately as long as three-quarters of propodus. tive length of the dactyli of the walking legs; these are shorter Dactylus of Mxp3 styliform or spatulate, inserted at or near (clearly less than half as long as the propodus) and more curved midlength of ventral margin of propodus, apex extending to in P. pisum (see Figure 4A in Becker & Türkay, 2010 and Figures or overreaching end of propodus ...... 7B, F–I & 8C–E in present study). In addition, in soft females of ...... Nepinnotheres pinnotheres P. pisum, the dactyli are of almost equal length on all walking legs (see Figure 5 in Becker & Türkay, 2010), while in the new species Carapace and pereiopods smooth. P2–P5 with short dactylus. the dactyli is clearly longer on the third pereiopod (Figure 7G). In Dactylus of Mxp3 styliform, inserted at base of ventral margin of addition, the carapace in P. pisum is subglobular (see Figure 5 in propodus, apex not extending to end of propodus ...... 4

Türkay, 2010, Figure 9F in present study) than in P. bicristatus (Figure 9G). The first gonopod of A. monodi was not described when the species was described by Manning (1993a), because the only male specimen lacked these appendages; therefore, this is the first time that the gonopod of A. monodi has been illustrated (Figure 9C).

About the specific host and associated pinnotherid species. Until now, only four species of pinnotherid crabs have been reported in association with bivalves of the genus Anomia (see Schmitt et al., 1973). Pinnotheres gordoni Shen, 1932, with distribution in China and Japan (Silas & Alagarswami, 1967), has been reported as living inside Anomia cytaeum Gray, 1850 (syn. Anomia lischkei Dautzenberg & Fischer, 1907), which has a general distribution in the waters of China, Japan and India (Higo et al., 1999; Souji & Radhakrishna, 2015). According to the description by Shen (1932), P. gordoni has pubescent chelipeds and walking legs, P4 being the longest pereiopod, while the dactylus of P5 is subequal to the propodus of the same leg. In these characters, it is clearly different from P. bicristatus. Two pinnotherid crabs from West Atlantic waters, namely Tumidotheres maculatus (Say, 1818), known to occur from Massachusetts to Argentina (Kruczynski, 1974), and Zaops ostreus (Say, 1817), present from Massachusetts to Cuba, Guadalupe and Brazil (Palacios et al., 2016), have been found inside Anomia simplex d’Orbigny 1853. This bivalve has a general distribution ranging from Nova Scotia to Mexico and the Caribbean (Rosenberg, 2009). Given that T. maculatus and Z. ostreus belong to different genera from that of Pinnotheres bicristatus sp. nov., the differences are major (see Campos, 1989; Manning, 1993a, 1993b). Among these differences to be noted are the size of the dactylus of P5, Fig. 9. Pinnotheres pectunculi: (A) soft female of San Roque, Cádiz, habitus in dorsal which is longer than the dactyli of P2–P4 in T. maculatus and Z. view (left) and details of left walking legs (right); (B) soft female from Marbella, Málaga, habitus in dorsal view and details of propodus and dactylus of right walking ostreus;inP. bicristatus sp. nov., P3 has the longest dactylus. legs P3–P5. Schematic drawings of first gonopod of European pinnotherids (setae Finally, the fourth species reported as being hosted by Anomia is removed): (C) Afropinnotheres monodi (IEO-CD-ICMAN/2442, Gulf of Cadiz); (D) the European Pinnotheres pisum, which has a general distribution Pinnotheres pisum (from Barbate Cadiz, June1994, Manjón-Cabeza & García Raso, from Norway to the Mediterranean Sea and Canary Islands 1988); (E) Nepinnotheres pinnotheres (from Malaga, 1978 within Atrina pectinata); (F) Pinnotheres pectunculi (figure modified from Becker & Türkay, 2010); (G) Pinnotheres (Triay-Portella et al., 2018), and has been recorded twice in bicristatus sp. nov. (holotype IEO-CD-ICMAN/2419). Scale bars: (C–E), 1.0 mm; F (from Anomia ephippium (Miranda y Rivera, 1921; Delongueville & Becker & Türkay, 2010), 200 µm; G, 0.5 mm. Scaillet, 2002). This bivalve is distributed from Norway to Morocco, and Angola, including also the Mediterranean Sea 4. Carapace of soft females subglobular-rounded. Eyes visible on (MolluscaBase 2018, accessed through WoRMS). The oldest record dorsal view. Dactylus of walking legs (P2–P5) with short, in A. ephippium is one by Miranda y Rivera (1921), from Malaga curved tips, less than half as long as propodus. Fixed finger (Alboran Sea) at 30 m depth, which mentioned the presence of of chela (propodus of P1) with single tooth ...... two females of P. pisum hosted in A. ephippium, and a second ...... Pinnotheres pisum (and the most recent known to the authors) is one female reported in Rota (Gulf of Cádiz) by Delongueville & Scaillet (2002). Both locations are within the area sampled in the present study. Carapace of soft females subtrapezoidal. Small eyes. Walking – Unfortunately, we cannot confirm these identifications because the legs (P2 P5) slender with short, pointed, curved dactylus, consid- two specimens from Malaga (Miranda y Rivera, 1921)nolonger erably less than half as long as propodus. Fixed finger of chela existinthecollectionwheretheyweredeposited(IEO),andthe (propodus of P1) with a well-developed basal tooth followed by Pinnotheres pectunculi photograph of the female from Rota in Delongueville & Scaillet additional small ones ...... (2002) does not allow a correct identification. However, we have analysed a large amount of material of A. ephippium from the Gulf of All males of the European pinnotherid species can be distin- Cádiz and Málaga and the only pea crab found living in association guished by the morphology of the first gonopod (see with this bivalve is Pinnotheres bicristatus sp.nov.Onthisevidence, Figure 9C–G). Each first gonopod has a different degree of it is very likely that the records of P. pisum in A. ephippium by curvature, from that of A. monodi, which is practically straight Miranda y Rivera (1921) and Delongueville & Scaillet (2002)are andonlylightlycurvedattheapex(Figure 9C)tothatofN. actually P. bicristatus sp. nov. The present new crab species probably pinnotheres, which is almost L-shaped (Figure 9E); in has a wider distribution throughout European and African waters Pinnotheres spp., it has an intermediate shape. The first gono- (North-east Atlantic and Mediterranean Sea), occurring wherever pod of P. pisum is straight and wide over almost its total length, A. ephippium is found. but its distal part is narrowed and curved (Figure 9D), while in With these two possible exceptions of Miranda y Rivera (1921) P. pectunculi and P. bicristatus sp. nov. the gonopod is curved and Delongueville & Scaillet (2002), Pinnotheres bicristatus sp. along its full length; however, the curvature of gonopod is more nov. had not been previously recorded in European waters. This pronounced in P. pectunculi (Figures 6C, D in Becker & oversight of its presence could be explained if P. bicristatus sp.

nov. is not native to the region of study and its introduction into October 2017 (3.3% of hosts infested) and February 2018 European A. ephippium had occurred recently. However, the rela- (26.7% infested). A positive relationship between crab prevalence tive high abundance of P. bicristatus in A. ephippium from the and the proportion of multiple infested hosts has been found in Gulf of Cadiz and the distribution range presently known support other pinnotherid crabs (Asama & Yamaoka, 2009; Mena et al., instead the hypothesis that this new pinnotherid is a native spe- 2014; Pérez-Miguel et al., 2019). Thus, it is not possible to reject cies, whose presence has been omitted because its host A. ephip- the view that the increased multiple infestations may be partially pium, which is not a commercial species, has been rarely collected associated with the increase in the proportion of hosts infested by and, consequently, has not been well studied. Besides, morpho- P. bicristatus sp. nov. observed between October 2017 and logical and molecular results of this study suggest that P. bicrista- February 2018: on average, 55.6 vs 77.7% of host specimens tus sp. nov. is a native species since it is closely related to P. pisum infested, respectively. However, since heterosexual pairs were col- and P. pectunculi: it has been shown that the geographic distribu- lected more frequently than expected by chance, a more probable tion of species plays an important role in their phylogenetic rela- explanation of the increase of male-female pairs inside a single tionships (Cuesta et al., 2012), and it would be more difficult to host in February was the increase of encounters for mating just find this molecular proximity between this new pinnotherid and before starting the spring spawning period. Mating that takes the other two native species if the former was an introduced place inside the hosts, where soft female crabs remain stationary, exotic species. For this reason, molecular data would support an and with males visiting various bivalves hosting soft females, has Atlantic-Mediterranean origin of P. bicristatus sp. nov. been suggested for other pinnotherid species (Soong, 1997; Ocampo et al., 2012). Pinnotheres bicristatus sp. nov. population hosted by Anomia Females of Pinnotheres bicristatus reached larger sizes than ephippium males, a sexual dimorphism that is very common among pea The high prevalence of Pinnotheres bicristatus sp. nov. in the crabs (e.g. see Soong, 1997; Ocampo et al., 2012;Drakeet al., common saddle oyster Anomia ephippium populations of the 2014). Concerning the relationship between host size and crab Gulf of Cádiz suggests that this crab forms a well-established size, a strong correlation was found between Anomia ephippium population in the area. Although the smallest sex-indeterminate size and ovigerous female size of P. bicristatus sp. nov.; this sug- individuals of P. bicristatus were not found inside A. ephippium, gests that availability of space inside a host is a relevant factor in the presence of at least 20% of males, together with hard and determining the final size of the crab in its sedentary phase (i.e. reproductive females, in this host makes it possible that a stable soft females). Although a significant correlation between host population of this crab exists even without any additional host. size and male crab size was also found in the population studied, For other pinnotherid species, sex ratios biased towards a lower the correlation was weaker than for females. Furthermore, for proportion of males have been explained by possible predation each specific host size, male crabs were smaller than females when males leave their hosts to find mates (Trottier & Jeff, hosted by it (Figure 3B). A similar sex-dependent pattern in the 2012). However, it is also plausible that other bivalve species relationship between host size and the symbiotic/parasitic crab were facultative hosts of males and of smaller specimens that size has been found previously for other pinnotherid species were not found inside A. ephippium, as is the case for (Soong, 1997; Kane & Farley, 2006;Ocampoet al., 2012; Afropinnotheres monodi found in the same area (Drake et al., Drake et al., 2014). As stated by Soong (1997), there is a closer 2014; Pérez-Miguel et al., 2019). The mussel Mytilus gallo- correlation in females than in males, between crab size and provincialis is the primary host of the soft (reproductive) females host size; this may be due to the longer association between of A. monodi, whereas other bivalves of small size (such as afemalecrabanditshost(femalesstaylongerinthesame Cerastoderma spp. and Scrobicularia plana) are the primary host than males do). Considering all the information available, hosts of hard females and males. Notwithstanding, about 15.8% we hypothesize that, initially, P. bicristatus sp. nov. hard females of A. monodi crabs hosted by mussels were males (Drake et al., donotmaketheirselectionofhostbysize(thereisnocorrel- 2014; Pérez-Miguel et al., 2019). If males and hard females of ation between the size of a hard female and that of its host). P. bicristatus were using another bivalve species as primary However, after their metamorphosis to soft females, they cannot host, it would probably be the case that this unknown host species leave the host and their growth might be limited by the host size, is also subtidal, since we did not collect any specimens of this crab which explains the observed strong correlation in size between (Pérez-Miguel et al., 2018) during an exhaustive sampling of soft females of P. bicristatus sp. nov. and the specimens of intertidal bivalves along the coasts of the Iberian Peninsula in A. ephippium hosting them. In the case of the smaller and 2016 and 2017. more motile males, they might not be limited by host size but, Since the timing and duration of reproductive periods is influ- as they may change hosts frequently, larger males may avoid enced by water temperature and/or the light-dark cycle (Sastry, relatively small (growth-limiting) hosts and this behaviour 1983), it is reasonable to assume that Pinnotheres bicristatus sp. could explain the significant but low correlation in size found nov. has one single reproductive period throughout the known between hosts and male crabs. distribution area (Figure 1). Currently, the available information on this topic is the presence of larval stages (zoeae and megalo- Final remark pae) found in November 2013 (Marco-Herrero et al., 2018), one As previously commented, Pinnotheres bicristatus sp. nov. prob- ovigerous female found in July 1995 in the Gulf of Cádiz, and ably has a wider distribution throughout European and African one ovigerous female found in April 2017 in the Alboran Sea waters linked to the occurrence of its host Anomia ephippium. (this study). According to this information, P. bicristatus sp. However, due to the scarce studies on this bivalve species, the nov. would have a long reproductive period extending from spring existence of P. bicristatus sp. nov. could have been overlooked to autumn. Furthermore, there was a clear predominance of until now. Therefore, we would like to underline the importance reproductive females with well-developed gonads both in of studying the plankton-collected larvae in combination with October 2017 and in February 2018, whereas ovigerous females DNA analyses, to discover, as in the present case, hidden cryptic were absent, suggesting that a slowdown of the final gonadal new species or non-indigenous species introduced in new areas. development occurred during the cold period and that spawning does not take place until the water temperature starts to rise. Author ORCIDs. Jose A. Cuesta, 0000-0001-9482-2336; J. E. García Raso, Concerning crab pairs, there was a remarkable increase between 0000-0003-3092-9518

Acknowledgements. We are grateful to all participants in the ARSA and Kruczynski WL (1974) Relationship between depth and occurrence of pea MEDITS surveys run by the Spanish Institute of Oceanography (IEO), from crabs, Pinnotheres maculatus, in blue mussels, Mytilus edulis, in the vicinity which many of the samples come, particularly to their chief scientists of Woods Hole, Massachusetts. Chesapeake Science 15, 167–169. (Ignacio Sobrino and Cristina García). We want to thank Carlos Sánchez Linnaeus C (1767) Systema Naturae, Tome I. Pars II. Editio duodecima, refor- (ICMAN) for laboratory work. Thank are also due to Maria Eugenia mata. Holmiae. Laurentii Salvii, pp. 533–1327. Manjón-Cabeza for sharing specimens collected in Barbate samples. We Manjón-Cabeza ME and García-Raso JE (1998) Population structure and want to thank two anonymous referees, and especially to Peter Ng for his growth of the hermit crab Diogenes pugilator (Decapoda: Anomura: detailed corrections, comments and suggestions that clearly improved the Diogenidae) from the Northeastern Atlantic. Journal of Crustacean Biology manuscript, and Royston Snart for the linguistic revision of the English text. 18, 753–762. Manning RB (1993a) West African pinnotherid crabs, subfamily ‘ Financial support. This study was partially funded by the Spanish Ministerio Pinnotherinae (Crustacea, Decapoda, Brachyura). Bulletin du Museum ’ de Economía y Competitividad (MINECO), Plan Nacional I + D and the national d’Histoire naturelle, Paris 15, 125–177. European FEDER funds through project AFROBIV (CGL2014-53557-P), and Manning RB (1993b) Three genera removed from the synonymy of the Project PB92-0415, supported by the Spanish M.E.C, DGICYT funds. Pinnotheres Bosc, 1802 (Brachyura: Pinnotheridae). Proceeding of the Biological Society of Washington 106, 523–531. References Marco-Herrero E, Drake P and Cuesta JA (2018) Larval morphology and DNA barcodes as valuable tools in early detection of marine invaders: a Ahyong ST, Lowry JK, Alonso M, Bamber NR, Boxshall GA, Castro P, new pea crab found in European waters. Journal of the Marine Biological Gerken S, Karaman GS, Goy JW, Jones DS, Meland K, Rogers DC and Association of the United Kingdom 98, 1675–1683. Svavarsson J (2011) Subphylum Crustacea Brünnich, 1772. In Zhang ZQ Mena S, Salas-Moya C and Wehrtmann IS (2014) Living with a crab: effect of (ed.) Animal Biodiversity: An Outline of Higher-Level Classification and Austinotheres angelicus (Brachyura, Pinnotheridae) infestation on the condition Survey of Taxonomic Richness. Zootaxa 3148, 165–191. of Saccostrea palmula (Ostreoida, Ostreidae). Nauplius 22,151–158. Asama H and Yamaoka K (2009) Life history of the pea crab, Pinnotheres Miranda y Rivera A (1921) Algunos crustáceos de la colección del laboratorio sinensis, in terms of infestation in the bivalve mollusc, Septifer virgatus. biológico de Málaga. Boletín de Pescas 6, 179–205. Marine Biodiversity Records 2, e77. MolluscaBase (2018) Anomia ephippium Linnaeus, 1758. Accessed through Becker C (2010) European Pea Crabs — Taxonomy, Morphology, and Host- WoRMS. Available at http://www.marinespecies.org/aphia.php?p=taxdetails Ecology (Crustacea: Brachyura: Pinnotheridae) (Unpublished PhD &id=138748 (accessed 25 July 2018). Dissertation). Goethe-Universität, Frankfurt am Main. Ng PKL, Guinot D and Davie PJF (2008) Systema Brachyurorum. Part 1, an Becker C and Türkay M (2010) Taxonomy and morphology of European pea annotated checklist of extant Brachyuran crabs of the world. Raffles Bulletin crabs (Crustacea: Brachyura: Pinnotheridae). Journal of Natural History 44, of Zoology 17, 255–276. 1555–1575. Ocampo EH, Nuñez JD, Cledón M and Baeza JA (2012) Host specific repro- Campos E (1989) Tumidotheres, a new genus for Pinnotheres margarita Smith, ductive benefits, host selection behavior and host use pattern of the pin- 1869, and Pinnotheres maculatus Say, 1818 (Brachyura: Pinnotheridae). notherid crab Calyptraeotheres garthi. Journal of Experimental Marine Journal of Crustacean Biology 9, 672–679. Biology and Ecology 429,36–46. Campos E (1993) Systematics and taxonomic remarks on Pinnotheres muli- Palacios Theil E, Cuesta JA and Felder DF (2016) Molecular evidence for non- niarum Rathbun, 1918 (Crustacea: Brachyura: Pinnotheridae). Proceedings monophyly of the pinnotheroid crabs (Crustacea: Brachyura: Pinnotheroidea), of the Biological Society of Washington 106,92–101. warranting taxonomic reappraisal. Invertebrate Systematics 30,1–27. Campos E (2002) Two new genera of pinnotherid crabs from the tropical Pérez-Miguel M, Cuesta JA, Navas JI, García Raso JE and Drake P (2018) Eastern Pacific (Decapoda: Brachyura: Pinnotheridae). Journal of The prevalence and effects of the African pea crab Afropinnotheres monodi Crustacean Biology 22, 328–336. on the condition of the mussel Mytilus galloprovincialis and the cockle Cuesta JA, Drake P, Martínez-Rodríguez G, Rodríguez A and Schubart CD Cerastoderma edule. Aquaculture 491,1–9. (2012) Molecular phylogeny of the genera Palaemon and Palaemonetes Pérez-Miguel M, Drake P, García Raso JE, Mamán Menéndez L, Navas JI (Decapoda, Caridea, Palaemonidae) from a European perspective. and Cuesta JA (2019) European Pinnotheridae (Crustacea, Decapoda, Crustaceana 85, 877–888. Brachyura): species, distribution, host use and DNA barcodes. Marine de Wulfen FXLB (1791) Descriptiones zoologicae ad Adriatici littora maris Biodiversity (online), doi: 10.1007/s12526-017-0754-8. concinnatae. Nova Acta Physico-Medica Academiae Caesareae Leopoldino- Rosenberg G (2009) Malacolog 4.1.1: A Database of Western Atlantic Marine Carolinae Naturae Curiosorum 8, 235–359. Mollusca. Available at http//www.malacolog.org/. Delongueville C and Scaillet R (2002) Bivalves et Crustacés – illustrations Sakai K (2000) On the occurrence of three species of crabs on Shikoku Island, d’une association. NOVAPEX/Société 3,51–55. Japan, and a new species, Pinnotheres taichungae nov. spec., from Taiwan Drake P, Marco-Herrero E, Subida MD, Arias AM and Cuesta JA (2014) (Decapoda, Brachyura). Crustaceana 73, 1155–1162. Host use pattern of the pea crab Afropinnotheres monodi: potential effects Sastry AN (1983) Ecological aspects of reproduction. In Vernberg WB (ed.) on its reproductive success and geographical expansion. Marine Ecology The Biology of Crustacea: 8. Environmental Adaptations. New York, NY: Progress Series 498, 203–215. Academic Press, pp. 179–270. Estoup A, Largiadèr CR, Perrot E and Chourrout D (1996) Rapid one tube Schmitt WL, McCain JC and Davidson ES (1973) Fam. Pinnotheridae, DNA extraction for reliable PCR detection of fish polymorphic marker and Brachyura I: Decapoda I. In Gruner HE and Holthuis LB (eds) transgenes. Molecular Marine Biology and Biotechnology 5, 295–298.. Crustaceorum Catalogus. Den Haag: W. Junk, pp. 1–160. García Raso JE and Manjón-Cabeza ME (2002) An infralittoral decapod Scopoli JA (1763) Entomologia Carniolica Exhibens Insecta Carnioliae Crustacean community of southern Spain affected by anthropogenic distur- Indigena et Distributa in Ordines, Genera, Species, Varietates. Methodo bances. Journal of Crustacean Biology 22,83–90. Linnaeana. Vindobonae, Typis Joannis Thomae Trattner, 30 unpaginated Gourret P (1887) Sur quelques Crustacés parasites des Phallusies. Comptes Rendus pages., pp. 1–420, 1 page errata, 43 unnumbered plates with figs. 1–680. Hebdomadaires des Séances de l’Académie des Sciences, Paris 104,185–187. doi: https://doi.org/10.5962/bhl.title.34434 Gourret P (1888) Études zoologiques sur quelques Crustacés parasites des Shen CJ (1932) The brachyuran crustacea of North China. Zoologia Sinica 9, Ascidies. Bibliothèque de l’École des Hautes Études, Paris, Section des 1–320. Sciences naturelles 36,1–64. Silas EG and Alagarswami K (1967) On an instance of parasitisation by the pea- Hesse CE (1872) Observations sur des crustacés rares ou nouveaux des cotes crab (Pinnotheres sp.) on the backwater clam (Meretrix casta (Chemnitz)) de France. Annales des Sciences naturelles, 5 ser., Zoologie, Paris 15,1–50. from India with a review of the work on the systematics, ecology, biology Higo S, Callomon P and Goto Y (1999) Catalogue and Bibliography of the and ethology of the pea crabs of the genus Pinnotheres latreille.Proceedings Marine Shell- Bearing Mollusca of Japan: Gastropoda, Bivalvia, of the Symposium on Crustacea held at Ernakulam, 1965, Symposium Polyplacophora, Scaphopoda. Osaka: Elle scientific Publications. Series 2. Journal of the Marine Biological Association of India 3, 1161–1227. Kane K and Farley GS (2006) Body size of the endosymbiotic pea crab Soong K (1997) Some life history observations on the pea crab, Pinnotheres Tumidotheres maculatus: larger hosts hold larger crabs. Gulf and tsingtaoensis, symbiotic with the bivalve mollusc, Sanguinolaria acuta. Caribbean Research 18,27–33. Crustaceana 70, 855–866.

Souji S and Radhakrishna T (2015) New distribution record of anomiid Triay-Portella R, Pérez-Miguel M, González JA and Cuesta JA (2018) On the pres- species (Bivalvia: Anomiidae) from Vizhinjam, Kerala, southwest coast of ence of Pinnotheres pisum (Brachyura, Pinnotheridae) in the Canary Islands (NE India. International Journal of Science and Research 4, 1488–1492. Atlantic), its southernmost distribution limit. Crustaceana 91,1397–1402. Ŝtevčić Z (1990) Check-list of the Adriatic decapod crustacea. Acta Adriatica Trottier O and Jeffs AG (2012) Biological characteristics of parasitic 31, 183–274. Nepinnotheres novaezelandiae within a Perna canaliculus farm. Diseases Subida MD, Arias AM, Drake P, García Raso JE, Rodríguez A and of Aquatic Organisms 101,61–68. Cuesta JA (2011) On the occurrence of Afropinnotheres monodi Udekem d’Acoz C d’ (1989) Seconde note sur les Crustacés Décapodes de la Manning, 1993 (Decapoda: Pinnotheridae) in European waters. Journal of Bretagne. De Strandvlo 8, 166–205. Crustacean Biology 31, 367–369. WoRMS (2018) Pinnotheres Bosc, 1801. Available at http://www.marine Tamura K, Stecher G, Peterson D, Filipski A and Kumar S (2013) MEGA6: species.org/aphia.php?p=taxdetails&id=106976 (accessed 25 July 2018). Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology Zar JH (2010) Biostatistical Analysis, 5th Edn. Upper Saddle River, NJ: and Evolution 30, 2725–2729. Prentice-Hall/Pearson, 944 pp.